Development of immunoassay methods utilizing an antigen-antibody reaction is now being made on a global scale as an early detection method for new virus-based diseases such as AIDS and adult T-cell leukemia as well as various cancers. The methods are designed to detect an antibody or antigen itself utilizing the property of an antibody, which is prepared when virus or a like serving as an antigen has invaded into a living organism, to specifically react with the corresponding antigen (antigen-antibody reaction). As a micro-immunoassay method for this purpose, RIA (radioimmunoassay), EIA (enzyme-immunoassay), FIA (fluorescence-immunoassay), etc. have heretofore been used in practice. These methods use antigens or antibodies which are labeled with an isotope, an enzyme or a fluorescent substance in order to detect the presence of the corresponding antibodies or antigens, respectively, that react therewith.
Among them, RIA is to quantitatively determine the amount of the specimen which contributed to the antigen-antibody reaction by measuring the amount of the radio-activity of the isotope fixed for labeling. To date, only this method is capable of ultramicro measurement in the order of picogram. However, RIA needs special installment since it has to handle radioactive substances and there have heretofore been restrictions with respect to time and place for use and the like in view of the half-life period of the radioactive substances, disposal of wastes and the like. In addition, those methods which use enzymes or fluorescent substances are designed to confirm if the presence of antigen-antibody reactions and therefore they remain semi-quantitative and the detectability limit is in the order of nanogram. Therefore, it has been a demand for an immunoassay method which has a detection sensitivity in the same order as that of RIA but is free of restrictions when in use.
Examples of thus-far published methods in which a laser beam is used to detect the presence of antigen-antibody reaction include a method designed for detecting liver cancer, in which micro-particles of a plastic are provided with an antibody to AFP (alpha-faeto protein) and the change in the mass as the result of agglomeration between the plastic particles due to antigen-antibody reaction is monitored by the change in the scattering or transmission of a laser beam. Reportedly, the detection sensitivity of this method is 10.sup.-10 g, which is one hundred times higher than conventional laser methods, but still one hundred times less sensitive as that of RIA. Since the method utilizes the change in Brownian movement of antigens and antibodies in an aqueous solution, it is necessary to precisely control the temperature of the aqueous solution containing a specimen upon measurement, and the method is defective in that it is susceptible to influences from outside such as ambient temperature and vibration.
Also, there is an essential limitation in the improvement of the detection sensitivity of the conventional laser beam scattering measurement and a large amount of specimen is required since only a part of the aqueous solution in which the specimen is dispersed is irradiated. One approach proposed for obviating this defect is to use as a vessel capillary tubes having the same diameter as that of a laser beam in the laser beam scattering measurement with view to reducing time for measurement and minimizing the amount of a specimen used (U.S. Pat. No. 4,605,305). However, there is a problem in that the detection sensitivity is decreased due to disturbance of measurement by the scattered light from the walls of the capillary tubes.
Further, magnetic micro-particles are used in an attempt to facilitate the adjustment of a specimen. More particularly, magnetic micro-particles are used in the removal or separation of various components in liquid samples. For example, U.S. Pat. No. 4,018,886 and 3,970,518 disclose methods in which magnetic micro-particles are used to collect a specified protein, the protein is cut off from the magnetic micro-particles and the precipitate obtained is observed with one's eyes. Also, U.S. Pat. No. 3,933,997 discloses a method in which a radioactive label is concentrated onto a specimen using magnetic micro-particles. In addition, methods prevail in which magnetic micro-particles bearing a receptor capable of being specifically bound to a specimen, and a label substance are used to separate unused label substances (enzymes, radioactive substances, fluorescent substances, etc.) using magnetic force (WO86/04684 and EP-A-30087). However, although some improvement in the degree or accuracy of separation is observed, the overall degree of separation is unsatisfactory since measurement itself is made according to conventional method and is controlled by the accuracy of the method used.
Further, U.S. Pat. No. 4,219,335 uses the above-described magnetic micro-particles in order to magnetically check the presence of a specified specimen in a body fluid. More particularly, a body fluid sample is affixed to the surface of the particles coated with a receptor reagent having a specific reactivity to the specimen, and upon contacting with the surface an immuno-reagent which has a reactivity with the receptor reagent or a complex of the receptor reagent and the specimen, a substance labeled with a reactance label such as micro-particles of a magnetic substance is used as an immuno-reagent, which is applied to the surface of a suspension of the specimen, and then, the change in the electrical reactance such as dielectric constant, conductivity or magnetic permeability of the surface is measured after removing unused immuno-reagent from the surface. This method, in which a measurement is made in order to directly check the presence or abundance of labeled micro-particles, is different at all in its principle from methods in which a laser beam is radiated into a measurement system including an immunocomplex labeled with micro-particles of the magnetic substance and corresponding outgoing light is optically measured.
The present inventors have previously investigated methods for detecting magnetic-labeled specimen using a magnetic head with high sensitivity and applied Japanese Patent Application No. Sho-62-104066 entitled "Detector for magnetic substances" and Japanese Patent Application No. Sho-62-137988 entitled "Magnetic immunoassay and apparatus therefor". However, the detection sensitivity thereof remains in the order of 1.times.10.sup.-9 g, which is by thousands times as sensitive as that of the laser magnetic immunoassay according to the present invention even when these methods of improving the detection sensitivity invented by the present inventors are applied.